Currently used biomarkers of tobacco smoke exposure have several disadvantages, including that they reflect short-term exposure and can therefore be affected by day-to-day variations. The aim of this study was to assess the validity of toenail nicotine levels as a biomarker of exposure to tobacco smoke for use in epidemiological studies. Toenails were collected in 1982 from 62,641 women participating in the Nurses' Health Study, whereas questionnaire data at that time provided information on active and passive smoke exposure. A stratified random sample of stored toenails from 106 women were selected according to their reported exposure category. Toenails were analyzed for nicotine levels by high-performance liquid chromatography. Toenail nicotine levels differed significantly according to tobacco smoke exposure (P < 0.0001). Among nonactive smokers, there was a significant difference in toenail nicotine levels between passive smokers (mean = 0.28 ng/mg) and nonexposed women (mean = 0.08 ng/mg; P = 0.0006). Among active smokers, there was also a significant difference (P = 0.04) in mean nicotine levels according to categories of cigarettes smoked (means for smokers of 1-14, 15-24, and 25 or more cigarettes/day were 0.94, 1.81, and 2.40 ng/mg). An overlap of the distribution of nicotine levels among passive and active smokers was found. Among all women, the correlation between nail nicotine levels and smoking exposure categories was r = 0.80 (P < 0.0001). The results of this study indicate that toenail nicotine level measurement is a valid biomarker for assessment of active and passive exposure to tobacco smoke. Nail nicotine levels may reflect aspects of active and passive exposure not captured by standard questionnaires and, thus, have the potential to provide better assessment of associations with health risk.